Prostate cancer encompasses a biological continuum ranging from a slow growing, indolent tumor to a highly aggressive and potentially fatal form. Most patients with metastatic prostate cancer receive drugs that block production of androgen, which promotes cancer cell proliferation. These drugs rarely cure cancer because the initial response is almost always followed by a relapse to an unresponsive, hormone-refractory stage (hormone refractory prostate cancer or HRPC). The relatively slow tumor progression coupled with the advancing age of the patients means that competing causes of death are substantive contributors to mortality in patients with prostate cancer. Thus while in HRPC patients tumor growth can be rapid and is a significant cause of death, in androgen dependent cancers it may be sufficient to slow down the progression to the more aggressive (HRPC) form.
Tumors stimulate the formation of new blood vessels (angiogenesis) in order to supply nutrients and oxygen to their structure. These vessels are formed rapidly without the controls of normal tissue and so exhibit various defects including leakiness, fragility, structural disorganization, aberrant morphology including large diameter and tortuosity, interrupted flow patterns, etc. The leakiness of the tumor vasculature is thought to be due in part to the abnormal expression of growth factors by tumor cells, and contributes to tumor growth and metastasis.1 The vessels have been shown to leak proteins such as fibrin, which forms a fibrinogen platform for stromal growth and further vessel development. A reduction in these abnormal vessels would result in a less favorable environment for tumor progression and an increase in the relative percentage of normal vessels to the area would lead to a more normal blood flow pattern to aid delivery of therapeutic agents.
The establishment of a prostate carcinoma primary cell line has thus far been unsuccessful (van Bokhoven A, Varella-Garcia M, Korch C, et al. Molecular Characteristics of Human Prostate Cell Lines. The Prostate (2003) 57:205-225). Cells from prostate tissue have proven to be among the most difficult from which to establish robust cultures. The cell lines PC-3 (ATCC #CRL1435), LNCaP (ATCC #CRL-1740), and DU145 (ATCC #HTB-41) are the classical cell models, all with metastatic sites of origin shown below:
AndrogenCell LineTissue of OriginSensitivityPC-3Adenocarcinoma; boneNometastasis.LNCaPAdenocarcinoma;Yessupraclavicular lymph nodemetastasis.DU145Carcinoma; brainNometastasis.
Human prostate cancer cell lines reflect the clinical range with LNCaP cells derived from a supraclavicular lymph node metastasis being androgen sensitive and representing the early forms and PC-3 cells derived from a bone metastasis being androgen insensitive and representing HRPC.
The use of radiopharmaceuticals (e.g., diagnostic imaging agents, radiotherapeutic agents) to detect and treat cancer is well known. In more recent years, the discovery of site-directed radiopharmaceuticals for cancer detection and/or treatment has gained popularity and continues to grow as the medical profession better appreciates the specificity, efficacy and utility of such compounds.
These newer radiopharmaceutical agents typically consist of a targeting agent connected to a metal chelator, which can be chelated to (e.g., complexed with) a diagnostic metal radionuclide such as, for example, technetium or indium, or a therapeutic metal radionuclide such as, for example, lutetium, yttrium, or rhenium. The role of the metal chelator is to hold (i.e., chelate) the metal radionuclide as the radiopharmaceutical agent is delivered to the desired site. A metal chelator which does not bind strongly to the metal radionuclide would render the radiopharmaceutical agent ineffective for its desired use since the metal radionuclide would therefore not reach its desired site. Thus, further research and development led to the discovery of metal chelators, such as that reported in U.S. Pat. No. 5,662,885 to Pollak et. al., hereby incorporated by reference, which exhibited strong binding affinity for metal radionuclides and the ability to conjugate with the targeting agent. Subsequently, the concept of using a “spacer” to create a physical separation between the metal chelator and the targeting agent was further introduced, for example in U.S. Pat. No. 5,976,495 to Pollak et. al., hereby incorporated by reference.
The role of the targeting agent, by virtue of its affinity for certain binding sites, is to direct the diagnostic agent, such as a radiopharmaceutical agent containing the metal radionuclide, to the desired site for detection or treatment. Typically, the targeting agent may include a protein, a peptide, or other macromolecule which exhibits a specific affinity for a given receptor. Other known targeting agents include monoclonal antibodies (MAbs), antibody fragments (Fab's and (Fab)2's), and receptor-avid peptides. Donald J. Buchsbaum, “Cancer Therapy with Radiolabeled Antibodies; Pharmacokinetics of Antibodies and Their Radiolabels; Experimental Radioimmunotherapy and Methods to Increase Therapeutic Efficacy,” CRC Press, Boca Raton, Chapter 10, pp. 115-140, (1995); Fischman, et al. “A Ticket to Ride: Peptide Radiopharmaceuticals,” The Journal of Nuclear Medicine, vol. 34, No. 12, (December 1993). These references are hereby incorporated by reference in their entirety.
In recent years, it has been learned that some cancer cells contain gastrin releasing peptide (GRP) receptors (GRP-R) of which there are a number of subtypes. In particular, it has been shown that several types of cancer cells have over-expressed or uniquely expressed GRP receptors. For this reason, much research and study have been done on GRP and GRP analogues which bind to the GRP receptor family. One such analogue is bombesin (BBN), a 14 amino acid peptide (i.e., tetradecapeptide) isolated from frog skin which is an analogue of human GRP and which binds to GRP receptors with high specificity and with an affinity similar to GRP. Bombesin and GRP analogues may take the form of agonists or antagonists.
Binding of GRP or BBN agonists to the GRP receptor increases the rate of cell division of these cancer cells and such agonists are internalized by the cell, while binding of GRP or BBN antagonists generally does not result in either internalization by the cell or increased rates of cell division. Such antagonists are designed to competitively inhibit endogenous GRP binding to GRP receptors and reduce the rate of cancer cell proliferation. See, e.g., Hoffken, K.; Peptides in Oncology II, Somatostatin Analogues and Bombesin Antagonists (1993), pp. 87-112. For this reason, a great deal of work has been, and is being pursued to develop BBN or GRP analogues that are antagonists. E.g., Davis et al., Metabolic Stability and Tumor Inhibition of Bombesin/GRP Receptor Antagonists, Peptides, vol. 13, pp. 401-407, 1992.
In designing an effective compound for use as a diagnostic or therapeutic agent for cancer, it is important that the drug have appropriate in vivo targeting and pharmacokinetic properties. For example, it is preferable that for a radiopharmaceutical, the radiolabeled peptide have high specific uptake by the cancer cells (e.g., via GRP receptors). In addition, it is also preferred that once the radionuclide localizes at a cancer site, it remains there for a desired amount of time to deliver a highly localized radiation dose to the site.
Moreover, developing radiolabeled peptides that are cleared efficiently from normal tissues is also an important factor for radiopharmaceutical agents. When biomolecules (e.g., MAb, Fab or peptides) labeled with metallic radionuclides (via a chelate conjugation), are administered to an animal such as a human, a large percentage of the metallic radionuclide (in some chemical form) can become “trapped” in either the kidney or liver parenchyma (i.e., is not excreted into the urine or bile). Duncan et al.; Indium-111-Diethylenetriaminepentaacetic Acid-Octreotide Is Delivered in Vivo to Pancreatic, Tumor Cell, Renal, and Hepatocyte Lysosomes, Cancer Research 57, pp. 659-671, (Feb. 15, 1997). For the smaller radiolabeled biomolecules (i.e., peptides or Fab), the major route of clearance of activity is through the kidneys which can also retain high levels of the radioactive metal (i.e., normally >10-15% of the injected dose). Retention of metal radionuclides in the kidney or liver is clearly undesirable. Conversely, clearance of the radiopharmaceutical from the blood stream too quickly by the kidney is also undesirable if longer diagnostic imaging or high tumor uptake for radiotherapy is needed.
Subsequent work, such as that in U.S. Pat. No. 6,200,546 and US 2002/0054855 to Hoffman, et. al, hereby incorporated by reference in their entirety, have attempted to overcome this problem by forming a compound having the general formula X-Y-B wherein X is a group capable of complexing a metal, Y is a covalent bond on a spacer group and B is a bombesin agonist binding moiety. Such compounds were reported to have high binding affinities to GRP receptors, and the radioactivity was retained inside of the cells for extended time periods. In addition, in vivo studies in normal mice have shown that retention of the radioactive metal in the kidneys was lower than that known in the art, with the majority of the radioactivity excreted into the urine.
New and improved radiopharmaceutical and other diagnostic compounds which have improved pharmacokinetics and improved kidney excretion (i.e., lower retention of the radioactive metal in the kidney) have now been found for diagnostic imaging and therapeutic uses. For diagnostic imaging, rapid renal excretion and low retained levels of radioactivity are critical for improved images. For radiotherapeutic use, slower blood clearance to allow for higher tumor uptake and better tumor targeting with low kidney retention are critical. Furthermore, for therapeutic use, a reduction in tumor vasculature, i.e. abnormal vessels, would result in a less favorable environment for tumor progression and an increase in the relative percentage of normal vessels to the area would lead to a more normal blood flow pattern to aid delivery of therapeutic agents.